Healthy bones are in a delicate balance of bone resorbing and bone forming processes in order to adapt the skeleton to the changing demands during the whole life span of an individual. This allows the skeleton to grow during childhood, as visualized by the growing skull where minerals are deposited on the outer side and bone is resorbed on the inner side giving space for the growing brain. During adulthood, bones can reinforce themselves for the adaption to loads when performing sports or carrying heavy weights. Bone remodeling is a process with a typical sequence: bone-resorbing osteoclasts dock on the surface of a distinct area and start resorbing bone material. As a result, lacunas or pits are formed. Onto such pits, bone-forming osteoblasts append and these cells deposit new bone material. At the end of such a cycle a region with a stronger bone structure remains. However, in older age or in females after menopause the equilibrium is shifted towards a net bone loss. Bone loss occurs also during long bed rest or under conditions of reduced gravity. As FIG. 1 illustrates, bone mass peaks in mid-age and then declines slowly. In women at risk accelerated bone loss occurs after menopause when estrogen hormone production ceases. Such bone loss is typically diagnosed as Osteoporosis or as Osteopenia (the milder preceding form), wherein the disease Osteoporosis has become a major health problem in populations with increasing life expectancies, particularly in western civilizations.
Not only humans, but also animals suffer from diseases of impaired bone formation both in older age or in young animals during periods of intensive growth. As an example, in animals, particularly in rapidly growing and food producing animals, such as poultry, leg weakness and, as one consequence, Tibial Dyschondroplasia (TD), is a major problem leading to losses and inferior meat quality. Also, due to reasons of animal welfare, the problem of Tibial Dyschondroplasia has to be avoided (see publication Tibial Dyschondroplasia, a Poultry Leg Problem. Rath NC, USDA/ARS, Poultry Production and Product Safety Research, Poultry Science Center, University of Arkansas, USA (Sep. 4, 2003)). Furthermore, laying hens have a massive turnover of calcium because of their eggshell production. Every day, approximately one tenth of the animals' calcium pool has to be taken up from nutrition, stored into the bones and mobilized within a few hours during production of the shell around the egg. Suboptimal supply of Vitamin D can, therefore, also lead to osteoporosis in laying hens.
Many approaches have been suggested and are pursued to prevent age-related and post-menopausal bone loss, wherein e.g. physical training and pharmaceutical treatment are the methods of choice for osteoporosis therapy. Today pharmaceutical treatment/drug treatment typically follows a therapeutic or curative approach, wherein therapy is started when a pathological condition has already been diagnosed, i.e. when bone mass or bone density has fallen under a certain minimum level of necessary bone mass or bone density, or, in the worst case, when a fracture has already occurred. Thus, prevention of an under-run of a minimum of necessary bone mass or bone density would be more preferable to therapy, but should start when bone density is still on a high level and is to be carried out for a long time, e.g. as illustrated in FIG. 1. Thereby, treatments which support physiological mechanisms and which are of natural origin may be a valuable alternative to curative treatment with synthetic drugs.
As of today, anti-osteoporotic drugs typically can be classified according to their mode of action into anti-resorptive agents, anabolics or steroid hormones, bone-forming agents and others, respectively. Alternatively or additionally, anti-osteoporotic drugs may be classified according to their chemical structures, which typically follow one of the above modes of action. Known anti-osteoporotic drugs include e.g. bisphosphonate groups, synthetic estrogens, Vitamin D and its metabolites, etc.
Today most prescribed drugs belong to the bisphosphonate group, acting on the bone resorbing osteoclasts and thereby reducing bone resorption (see e.g. Fleisch H. et al., Endocrine Reviews (1998)19(1): 80-100 Bisphosphonates: Mechanisms of Action). A disadvantage of treatment with drugs of the bisphosphonate group is the blockage of bone-turnover and thus a reduction of bone remodeling.
As evidenced by the strong loss of bone mass after menopause, female sex hormones also possess a strong effect on bone. Therefore, hormone replacement therapy with synthetic estrogens may represent an effective Osteoporosis treatment. Such treatment, however, limited to females and is no longer recommended today since a large clinical study showed an increased incidence of breast cancer as an adverse reaction.
A further; more physiological and thus more promising approach is the support of the Calcium regulation and thus of the natural bone mineral household of the patient to be treated, either as a prevention or as a therapy for the above diseases.
There are many factors, which are involved in Calcium regulation and the natural bone mineral household. Agents acting on Calcium homeostasis include e.g. hormones, such as the hormones calcitonin or parathyroid hormone and synthetic derivatives thereof. Such peptide hormones, however, cannot be administered orally, but need to be administered by injection or as a nasal spray, and thus do not allow an easy administration of the drugs to a patient in need thereof.
The most important factor of the Calcium regulation in animals and humans, including the natural bone mineral household, involves Calcium, typically in the form of a preferably soluble salt or as Ca2+. Additionally to the above, Calcium also represents a key agent in intra-cellular signaling, nerve impulse transmission and muscle contraction (Cashman et al., Novartis Found Symp. 2007, 282:123-38, discussion 138-42, 212-8; and Parfitt A M. Bone. 1987, 8 Suppl. 1: S1-8). The insufficient provision of Calcium to the human or animal body may thus lead to concentrations resulting in an under-run of a minimum of necessary bone mass or bone density. On the other hand, without an effective regulation, the content of Calcium in the human or animal body may reach too high concentrations which interfere with intra-cellular signaling, nerve impulse transmission and muscle contraction, or which may lead to toxic side effects. Therefore, in all warm-blooded animals, a tight regulation system prevents the body from toxic calcium concentrations.
A further main component of Calcium regulation in the natural bone mineral household is Vitamin D. Vitamin D is an essential nutrient for optimal bone development as seen in the prevention and healing of rickets, wherein natural and non-natural derivatives of Vitamin D metabolites are known to be used. However, naturally occurring Vitamin D in the form of cholecalciferol (Vitamin D3) or ergocalciferol (Vitamin D2) is biologically not active. Accordingly, naturally occurring Vitamin D is not able to cure slowly developing bone diseases such as Osteoporosis as has been stated in a consensus conference (Osteoporosis Prevention, Diagnosis, and Therapy. NIH Consens Statement Online 2000 Mar. 27-29; 17(1): 1-36.). This is due to the fact that naturally occurring Vitamin D requires two conversion steps to be active, whereby the second step is tightly regulated. According to a first step, after formation in the skin or uptake by ingestion, cholecalciferol (Vitamin D3) is converted in the liver of man and animal into its storage form 25-hydroxyvitamin D3 (also abbreviated as 25(OH)D3). A full Vitamin D3 store protects a person for 2 to 3 months against rickets. The storage form is, however, biologically still not active—it needs activation in a second step to the active form of Vitamin D3, i.e. 1,25-dihydroxyvitamin D3 (also abbreviated as 1,25(OH)2D3, Calcitriol), by a kidney enzyme. The active form 1,25-dihydroxyvitamin D3 then activates a gene product in the sensitive tissue. In intestine this is Calbindin, the Calcium-binding protein which is finally capable to take up Calcium from food. The so formed active metabolite 1,25-dihydroxyvitamin D3 furthermore controls Calcium uptake in the intestinal tract and its deposition into and mobilization from bone.
Nevertheless, such natural control mechanisms do most often not lead to a sufficient concentration of the active metabolite in the human or animal body to prevent slow developing bone diseases such as Osteoporosis, particularly in old age, or to prevent other diseases in animals, such as Tibial Dyschondroplasia in poultry.
Since the first discovery of the active principle, various approaches have been started to provide Vitamin D3 for use in medicine, since Vitamin D3 is not abundantly present in nutrition; only marine oils contain substantial amounts. Provision of Vitamin D3 or its metabolites, thus represents an essential basis and also a challenging aspect in efficient therapy of any of the diseases mentioned above. E.g., U.S. Pat. No. 5,508,392 discloses the use of synthetic Vitamin D3 glycosides, Vitamin D3 orthoester glycosides, Vitamin D3 analog glycosides and Vitamin D3 analog orthoester glycosides for the treatment of osteoporosis.
A negative drawback of the administration of Vitamin D3 and its synthetic analogs is the particularly narrow therapeutic window for medication and the risk of hypercalcemia, i.e. an abnormally and toxic high blood concentration of Calcium, which can eventually cause severe damage to soft tissues and kidneys. It may be caused by intoxication with Vitamin D3 (hypervitaminosis D3) due to overdoses at more than 100 times (of) the recommended daily allowance. Such an essential drawback is in particular known for 1,25-Dihydroxyvitamin D3, wherein a small window for medication is open in the range between the effective dose and the dose with beginning toxic side effects, the rate of which is only 2 to 5. As an example, a measurable adverse effect of high doses of 1,25-Dihydroxyvitamin D3 was observed in poultry production leading to a remarkably lower weight gain. However, regardless of the above mentioned drawback, Vitamin D3 and its analogs still appear to be an attractive compound for use in any of the above therapies.
As to whether a therapy is suitable nonetheless also depends on further factors, e.g. the costs for preparing the active compounds used therein. The bottle neck for a cost efficient therapy of diseases as mentioned above using Vitamin D3 or its analogs is, consequently, a cheap provision or preparation of Vitamin D metabolites.
According to one possibility, Vitamin D metabolites may be provided by chemical stereoselective synthesis. Unfortunately, stereoselective synthesis is typically labor intensive and requires many synthesis and purification steps to obtain an enantiomerically enriched or even pure form of a Vitamin D3 or its metabolities. Accordingly, stereoselective synthesis of Vitamin D is expensive and does not allow a cost efficient therapy of any of the diseases mentioned above at the present stage, even though many synthetic Vitamin D3 medicaments are admitted and often prescribed. Furthermore, synthetically produced 1,25-Dihydroxyvitamin D3, administered in high concentrations, has the above mentioned drawback of a particularly narrow therapeutic window for medication and entails the risk of hypercalcemia. Thus, other sources may be preferred for provision of Vitamin D3 or its metabolites.
Still, it was a surprise when plants were discovered that contain Vitamin D metabolites in high amounts. Plants of this type include e.g. species of the family of Solanacea (solanaceous herbs), particularly Solanum glaucophyllum (also termed Solanum malacoxylon or Solanum glaucum), Solanum torvum, Solanum esuriale, Solanum verbascifolium, Cestrum diurnum, etc., the species Nierembergia veichtii, and species from the family of Gramineae, particularly Trisetum flavescens, etc. Intensive research in the last decades furthermore revealed that leaves of tomato plants (Lycopersicon esculentum from the family of Solanacea) exhibit a certain amount of Vitamin D3 in the form of 25(OH)D3 and 1,25(OH)2D3-glycosides (Prema and Rhagamulu, 1996, Phytochem. 42(3), 617-620). Similarly, potato plants (Solanum tuberosum), aubergine plants (Solanum melongena) and courgette plants (Cucurbita pepo L. ssp. pepo convar. giromontina) (see e.g. Aburjaj et al. 1998, Phytochem. 46(6), 1005-1018) as well as Nicotiana glauca (blue green tobacco from the family of Solanaceae) (Skliar et al, 2000, Plant Science 156, 193-199) showed a considerable amount of Vitamin D. Thus, there are potentially some plants, which may serve as a basis for provision of Vitamin D or its metabolites.
Basis for this discovery was the occurrence of beneficial effects upon feeding animals with leaves or other parts of these plants. Several of these beneficial effects of dried leaves of such calcinogenic plants have been published (see e.g. Boland et al., Plant Science 00, 1-13 (2003)). Also, some applications disclosed the use of extracts from these plants. E.g. U.S. Pat. No. 5,776,461 discloses cosmetic compositions containing phytovitamin D, particularly natural skin care composition containing selected hydroxylated Vitamin D compounds or their glycosides, which are derived from plant sources (phytovitamins D).
One plant with the highest concentrations of Vitamin D turned out to be Solanum glaucophyllum. In particular, it has been found that Solanum glaucophyllum, earlier known as Solanum malacoxylon, contains Vitamin D. In parts of the species Solanum glaucophyllum the active component was identified as the Vitamin D3 metabolite 1,25-Dihydroxyvitamin D3 (see e.g. De Vernejou) et al., La Nouvelle Presse medicate, 7, 22, 1941-43 (1978)).
While extensive literature exists on the Vitamin D active components in Solanum glaucophyllurn, very little is known on other components of the plant. Solely one publication described the presence of the alkaloid solasodine in the plant (see Jain and Sahoo, Pharmazie (1986) 41:820-821) and one publication described the presence of phenolic compounds; however no quantitative portions of these compounds were given (Rappapport et al., Phytochemistry (1977) 16:1115-6). Apart from its main component 1,25-dihydroxyvitamin D3, in Solanum glaucophyllum was found a series of phenolic glycosides by butanol extraction of the plant including hydroquinone, kaempferol and quercetin and the following known glycosides arbutin, O-methylarbutin, isoquercetin, avicutarin, rutin, kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside. A new quercetin trioside, the compound quercetin 3-O-[2G-b-D-apiosyllrutinoside was also found in Solanum glaucophyllum. Many of these phenolic compounds are constituents of all plants and no quantitative content was given in the publication of Rappaport et al. (1977, supra). Furthermore, flavonoids, a subclass of plant phenols, are typically discussed as antioxidants, whereas a positive activity in bone formation has not been shown or discussed. Moreover, only for (the plant phenolic sub-class) flavonols (quercetin and kaempferol) an effect on bone cells has been described. For example, recent in vitro experiments indicate an inhibition of osteoclast formation and differentiation of osteoctastic precursor cells by quercetin and rutin. Yamaguchi found a potent inhibitory effect on osteoclastogenesis and bone resorption rather than bone formation in vitro by quercetin and kaempferol (Yamaguchi et al., Mol Cell Biochem. 2007 Jun. 1). On a genomic level quercetin and its glucuronide promote an increase of the mRNA level of bone sialoprotein (Kim et al., J Cell Biochem. 2007 Jun. 1). Other in vitro studies in cells of the osteoctastic lineage confirmed an inhibiting effect on osteoclast differentiation, a critical determinant step in bone resorption, but no positive effect on bone formation was shown (Wattel et al., J Cell Biochem. 2004 May 15; 92(2):285-95).
On a higher level, experiments in ossicle organ cultures (Sziklai and Ribari, Acta Otolaryngol. 1995 March; 1 1 5(2):296-9) and in rat calvarial osteoblast cells (Yang et al., Zhong Yao Cai. 2006 May; 29(5):467-70) also showed an inhibiting effect of quercetin on osteoblastic cells. Furthermore, in two studies with intact animals quercetin was effective in bone mineral metabolism, biomechanical strength and bone structure in streptozutocin-induced diabetic rats (Kanter et al., Cell Biochem Funct. 2007 Jan. 31) and rutin in ovariectomy-induced osteopenia in rats (Horcajada-Molteni et al., J Bone Miner Res. 2000 November; 15(11):2251-8. Comment in: J Bone Miner Res. 2001 May; 16(5):970-1). Nevertheless, even if these compounds have been determined to occur in plants of Solanum glaucophyllum, prior art only discusses Vitamin D as sole active principle in the treatment of diseases as mentioned above.
At the time of discovery of the high content of Vitamin D metabolite 1,25-dihydroxyvitamin D3 in Solanum glaucophyllum, it has been speculated whether the plant can be used to treat bone diseases in man and animals and the biological activity of the extract has been explored in laboratory animals. Such experiments used Vitamin D-depleted animals in order to prove the Vitamin D activity (see e.g. De Vernejoul. et al., (1978), supra). Azcona (Azcona et al., Zootechnica Intern. February 1982 p. 12-13). Morris (Morris K M L, The Veterinary Record, 101, 502-504 (1977)) found a higher eggshell strength after feeding dry leaves of Solanum glaucophyllum. Norrdin (Norrdin at al, Calcified Tissue International (1979) 28(1):239-243) noticed a higher bone mass and breaking strength in chicken bones after application of dry leaves of the same plant. An aqueous leaf extract furthermore showed a higher Vitamin D activity after incubation with rumen fluid of bovines and sheep (Mello and Habermehl, Dtsch Tierarztl Wochenschr. 1992 September; 99(9):371-6). From WO 85/05110 it is also known that extracts from the leaves of the South-American plant Solanum glaucophyllum contains 1,25-Dihydroxyvitamin D3 and a water-soluble principle which is different from 1,25-Dihydroxyvitamin D3 and which, upon treatment with rumen fluid, yields 1,25-Dihydroxyvitamin D3 plus a water-soluble carbohydrate In said prior art it is further stated that the water-soluble extract of Solanum glaucophyllum has a biological activity which is similar to that of 1,25-Dihydroxyvitamin D3. From the Austrian Patent Specification AT 398 372 B which was published approximately 9 years after WO 85/05110 it can be seen that dried leaves of Solanum glaucophyllum indeed have the alleged activity but also the above mentioned known drawback of high toxicity. Later, Cheng et al. (2004) (Cheng et al., Poult Sci. 2004 March; 83(3):406-13.) found improved phosphorus utilization in broiler chickens when fed leaves of Solanum glaucophyllum. Furthermore, Foote et al. (2004) (Foote et al., J Anim Sci. 2004 January; 82(1):242-9) found that Vitamin D and its metabolites obtained from plants containing such metabolites can improve meat tenderness when fed before slaughtering. However, all these attempts share the above mentioned known drawback of high toxicity.
Furthermore, dried leaves of the plant were given to patients suffering from hyperthyroidism and kidney insufficiency for up to 7 days. A normalizing effect on plasma calcium was observed (see e.g. Mautalen et al., Calcif Tissue Res. 1977 May; 22 Suppl:534-7; and Herrath et al., Vitamin D, Chemical and Clinical Aspects related to Calcium Metabolism. Pp. 703-708. W. de Gruyter, Berlin, Germany, 1977). Particularly limiting in this case is the application of an unpurified extract of unknown activity, which typically contains toxic alkaloids.
Common to all these trials was additionally that the material used was not characterized and no active component was measured in all experiments. Furthermore, no other than Vitamin D-like effects were described for Solanum glaucophyllum. 
On the other hand, several papers have been published on attempts to analyze the active principle of Solanum glaucophyllum and to prepare plant extracts For example, Peterlik and Wasserman (1975) (see Peterlik and Wasserman, FEBS Letters (1975) 56:16-19) extracted dry leaves with chloroform/methanol mixtures, while Mello and Habermehl (1998) extracted dry plant material with hot water (see Mello and Habermehl, Dtsch Tierarztl Wochenschr. 1998 January; 105(1):25-9). Further purification was performed by chromatography on Sephadex G1 5 and Sephadex LH2O (see Vidal et al., Turrialba (1985) 35:65-70) or by preparative HPLC chromatography on columns with silica and C18-modified silica as stationary phases (Skliar et al., J Steroid Biochem Mol Biol. 1992 December; 43(7):677-82).
The publication of von Rosenberg S. J., 2006 (Rosenberg S. J., 2006, PhD-thesis, Ludwigs-Maximilians-University, Munich, Germany) and the subsequent publication of von Rosenberg S. J. et al. 2007, (Rosenberg S. J. et al., The Journal of Steroid Biochemistry and Molecular Biology, Volume 103, Issues 3-5, March 2007, Pages 596-600) furthermore disclose plant extracts from Solanum glaucophyllum and Trisetum flavescens for use as food supplement or for human therapy, particularly Osteoporosis. The plant extracts disclosed therein were provided by Herbonis AG, Basel, Switzerland, and have been prepared by aqueous extraction of dried plants (leaves) followed by purification of the extract using high pressure liquid chromatography (HPLC) with Sephadex as a matrix material. The extract solely contained the active Vitamin D metabolite 1,25-Dihydroxyvitamin D3 as active principle.
Even if some laboratory extraction methods have been published in the meantime, none of the above publications likewise discloses a method for extracting and/or purifying a plant extract containing Vitamin D compounds with a sufficiently high, i.e. an industrially applicable, yield. The methods used are convenient to find the active principle 1,25-dihydroxyvitamin D3 but no attempt has been made to optimize yield and minimize toxic by-products. Extractions with pure water or aqueous compositions to separate the free 1,25-dihydroxyvitamin D3 from the bound form have been made, or, chloroform, an itself toxic solvent, was used to isolate the free 1,25-dihydroxyvitamin D3 from the water-soluble components. For the purification of the active principle 1,25-dihydroxyvitamin D3 several methods have been described using column chromatography with silica material or Sephadex gels, but such methods are not feasible for the production of larger quantities of extracts. Particularly, chromatography on silica material gives lower yields of Vitamin D metabolites and thus does not allow production of Vitamin D containing plant extracts in industrial scale, showing a considerable amount of Vitamin D. Furthermore, none of the above applications managed to provide a plant extract, which overcomes at least in part the above drawback of high toxicity when administered in higher concentrations.